Biochemical characterization of <i>Sp</i>MetAP1a.

<p>a) Enzyme binding curve of the Met-pNA at varied concentrations is depicted with the fit value of 0.97924. b) Metal dependency on the activity of the enzyme is tested using five different metal salts ranging from 1-1000 µM. Cobalt is identified as the best co-factor followed by manganese. In the presence of nickel, negligible activity was observed. Zinc and magnesium did not activate the enzyme. c) Activity dependency of the <i>Sp</i>MetAP1a on pH change. Maximum activity was observed at pH 7.5. d) Substrate specificity of the <i>Sp</i>MetAP1a. Among the eleven amino acid-<i>p</i>NA substrates tested, only methionine was hydrolyzed.</p

Inactive conformation of the <i>Sp</i>MetAP1a.

<p>Cartoon representation of the structural alignment of <i>Sp</i>MetAP1a (yellow) and <i>Ec</i>MetAP1a (red). Note that the β-hairpin loop collapses in to the active site there by the peptide with methionine on the amino terminus cannot bind to the enzyme.</p

Crystal structure of <i>Sp</i>MetAP1a.

<p>a) Cartoon representation of two molecules (brown and gray) in the asymmetric unit. Two inserts⁶³, insert and ¹⁰³insert are shown in red in one of the molecules. The overall pita-bread fold is conserved like in other known MetAPs. b) Stereo diagram of the two inserts on the top and the^198-211β-hairpin (equivalent region in <i>Ec</i>MetAP1a is 170-183 is shown in brown) undergoes a conformational change and occupies the active site where the substrate methionine side chain usually binds while the metal binding H199 moves away from the active site. The 2<i>F</i>o-<i>F</i>c electron density is shown at 1.2 σ. Two metal ions (blue spheres) and the product methionine (brown sticks) are shown from the <i>Ec</i>MetAP1a crystal structure. The black arrow shows the direction of the hairpin flip. The red arrow points to the histidine that is essential for the activity but flips away in the <i>Sp</i>MetAP1a structure (H199). c) Electron density cover (2<i>F</i>o-<i>F</i>c) of⁶³insert and ¹⁰³insert at 1.0 σ. The tight helix turn in <i>Ec</i>MetAP1a (brown helix) extends into a longer loop in the⁶³insert. Reverse β-turn is represented near 109-111 residues.</p

a) Representation of domain structure of sub-classes of MetAP based on the crystal structures.

<p>Common catalytic regions are shown in green. Type Ia has the basic structure required for catalysis. Type Ib and Type Ic have extra regions on the amino terminus. Type II MetAP has a 60 amino acid insert in addition to N-terminal extension (yellow). On the left of each bar is the representative crystal structure in cartoon diagram. Pink spheres in the middle indicate the two metal ions in the active site. b) Sequence alignment of Type I MetAPs from <i>S. pneumoniae</i> (Type Ia), <i>E. coli</i> (Type Ia), <i>M. tuberculosis</i> (Type Ic) <i>and </i><i>human</i> (Type Ib). Numbering and the secondary structure representation on the top of the alignment are based on the <i>Sp</i>MetAP1a crystal structure. Metal binding conserved residues are marked as asterisk. Two inserts are identified⁶³; insert and ¹⁰³insert. These inserts are predominantly present in the streptococcus bacterial Type Ia MetAP¹⁰⁹. DLNVSK and ¹²⁴KKYT are two sequences marked in red are predicted to have respectively glycosylation and phosphorylation sites. c) Phylogenetic tree of 24 Type I MetAPs from streptococcal and lactococcal bacteria that contain the two inserts observed in the Figure 1b. Note that each of these species forms separate clusters.</p

Identification of the Molecular Basis of Inhibitor Selectivity between the Human and Streptococcal Type I Methionine Aminopeptidases

The methionine aminopeptidase (MetAP) family is responsible for the cleavage of the initiator methionine from newly synthesized proteins. Currently, there are no small molecule inhibitors that show selectivity toward the bacterial MetAPs compared to the human enzyme. In our current study, we have screened 20 α-aminophosphonate derivatives and identified a molecule (compound <b>15</b>) that selectively inhibits the <i>S. pneumonia</i> MetAP in low micromolar range but not the human enzyme. Further bioinformatics, biochemical, and structural analyses suggested that phenylalanine (F309) in the human enzyme and methionine (M205) in the <i>S. pneumonia</i> MetAP at the analogous position render them with different susceptibilities against the identified inhibitor. X-ray crystal structures of various inhibitors in complex with wild type and F309M enzyme further established the molecular basis for the inhibitor selectivity

Identification, Biochemical and Structural Evaluation of Species-Specific Inhibitors against Type I Methionine Aminopeptidases

Methionine aminopeptidases (MetAPs) are essential enzymes that make them good drug targets in cancer and microbial infections. MetAPs remove the initiator methionine from newly synthesized peptides in every living cell. MetAPs are broadly divided into type I and type II classes. Both prokaryotes and eukaryotes contain type I MetAPs, while eukaryotes have additional type II MetAP enzyme. Although several inhibitors have been reported against type I enzymes, subclass specificity is scarce. Here, using the fine differences in the entrance of the active sites of MetAPs from Mycobacterium tuberculosis, Enterococcus faecalis, and human, three hotspots have been identified and pyridinylpyrimidine-based molecules were selected from a commercial source to target these hotspots. In the biochemical evaluation, many of the 38 compounds displayed differential behavior against these three enzymes. Crystal structures of four selected inhibitors in complex with human MetAP1b and molecular modeling studies provided the basis for the binding specificity